CN111266142A - Detection chip, modification method thereof and reaction system - Google Patents
Detection chip, modification method thereof and reaction system Download PDFInfo
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Abstract
The application discloses a detection chip, a modification method and a reaction system thereof, wherein the modification method comprises the steps of activating the detection chip with silicon oxide to convert the silicon oxide into silicon hydroxyl; and (3) placing the detection chip with the silicon hydroxyl in an organic solution of 3-glycerol propyl trimethoxy silane to obtain the detection chip with the surface combined with the epoxy group. A series of surface chemical reaction operations are carried out on the surface of the detection chip, so that the surface of the detection chip generates the epoxy group connected through chemical bonds, the problem that a compound film containing the epoxy group on the detection chip is easy to fall off in the related technology is solved, and the subsequent protein coupling efficiency is improved.
Description
Technical Field
The application relates to the technical field of biomedicine, in particular to a detection chip, a modification method and a reaction system thereof.
Background
The term microfluidic chip was originally derived from the micro total analysis system (μ TAS) proposed by Manz and Widmer in the 90 th of the 20 th century. Professor Manz successfully applies the MEMS technology to the field of analytical chemistry and realizes high-speed capillary electrophoresis on microchips in the near future, and the results are published in journal of Science and the like, and this field is rapidly receiving attention from the academia and becomes one of the leading Science and technology fields in the world. Lab-on-a-Chip (Lab-on-a-Chip) and Microfluidic Chip (Microfluidic Chip) are different names that have been proposed in this field, and as the application of this discipline expands from initial analytical chemistry to a number of research and application areas, and researchers's deep understanding of this discipline, Microfluidic chips have become a collective term for this area.
A biochip is a chip technology, which is characterized by orderly arranging a series of known recognition molecules on the surface of a substrate in a lattice manner, enabling the recognition molecules to be combined or reacted with a substance to be detected, displaying and analyzing the recognition molecules by a certain method, and finally obtaining information such as the chemical molecular structure of the substance to be detected. The biochip has wide application and may be used in molecular biology, biomedicine, medicine research and development and other fields. Compared with the traditional detection method, the method has the characteristics of high flux, high information content, rapidness, miniaturization, automation, wide application and the like.
Disclosure of Invention
The embodiment of the application provides a detection chip, a modification method thereof and a reaction system, aiming at providing a detection chip with a high-density epoxy distribution formed on the surface, which can be used for subsequent protein coupling.
The modification method for the detection chip provided by the embodiment of the application comprises the following steps:
activating the detection chip with the silicon oxide to convert the silicon oxide into a silicon hydroxyl group;
and (3) placing the detection chip with the silicon hydroxyl in an organic solution of 3-glyceryl propyl trimethoxy silane to obtain the detection chip with the surface combined with an epoxy group.
Optionally, in the above modification method provided in this embodiment of the present application, the detecting chip with the silicon hydroxyl group is placed in an organic solution of 3-glyceropropyltrimethoxysilane to obtain the detecting chip with an epoxy group bound to the surface, which specifically includes:
and (3) putting the detection chip with the silicon hydroxyl into a toluene solution of 0.5-5% (v/v) 3-glyceropropyl trimethoxy silane, and hermetically soaking for 24-72 h at the temperature of between room temperature and 70 ℃.
Optionally, in the modification method provided in this embodiment of the present application, the placing the detection chip having the silicon hydroxyl group into a toluene solution of 0.5% -5% (v/v) 3-glyceropropyltrimethoxysilane, and hermetically soaking the detection chip for 24h-72h at a temperature of between room temperature and 70 ℃, specifically including:
the detection chip with the silicon hydroxyl group is put into a toluene solution of 3% (v/v) 3-glyceropropyl trimethoxy silane and is hermetically soaked for 24 hours at 70 ℃.
Optionally, in the modification method provided in this embodiment of the present application, the activating treatment on the detection chip with silicon oxide to convert the silicon oxide into a silicon hydroxyl group specifically includes:
putting the detection chip with silicon oxide into the piranha solution, and soaking for 12-24 h at the temperature of 70-90 ℃; the piranha solution is composed of concentrated sulfuric acid and 30% hydrogen peroxide, wherein the volume ratio of the concentrated sulfuric acid to the 30% hydrogen peroxide is 1: 3.
Optionally, in the modification method provided in this embodiment of the present application, after each step is performed, the following processing is performed on the detection chip:
washing the detection chip twice by using deionized water;
and ultrasonically cleaning the washed detection chip in deionized water, and drying the detection chip by using nitrogen for later use.
Optionally, in the modification method provided in this embodiment of the present application, before the activation treatment of the detection chip with silicon oxide, the modification method further includes:
and sequentially adopting acetone, ethanol and deionized water to ultrasonically clean the detection chip with the silicon oxide, and adopting nitrogen to blow the cleaned detection chip for later use.
Optionally, in the modification method provided in this embodiment of the present application, before the performing ultrasonic cleaning on the detection chip with silicon oxide by using acetone, the method further includes:
and pretreating the glass substrate to form the detection chip with silicon oxide.
Optionally, in the modification method provided in the embodiment of the present application, the pretreating the glass substrate to form the detection chip having silicon oxide specifically includes:
forming a plurality of spotting platforms on the glass substrate;
and depositing a hydrophilic layer which is 300nm thick and is made of silicon oxide on the layer where the sample application platforms are located by adopting a plasma enhanced chemical vapor deposition method at the temperature of 390 ℃, etching the hydrophilic layer, and reserving the silicon oxide in the area where the sample application platforms are located to obtain the detection chip with the silicon oxide.
Based on the same inventive concept, the embodiment of the present application further provides a detection chip, including: the printing device comprises a glass substrate, a plurality of printing platforms positioned on the glass substrate, and a hydrophilic layer covering each printing platform, wherein the hydrophilic layer comprises silicon oxide with a chemical modification group; wherein the content of the first and second substances,
the chemical modification group is obtained by adopting the modification method.
Based on the same inventive concept, the embodiment of the application also provides a reaction system, which comprises the detection chip.
The beneficial effect of this application is as follows:
the embodiment of the application provides a detection chip, a modification method and a reaction system thereof, wherein the modification method comprises the steps of activating the detection chip with silicon oxide to convert the silicon oxide into silicon hydroxyl; and (3) placing the detection chip with the silicon hydroxyl in an organic solution of 3-glycerol propyl trimethoxy silane to obtain the detection chip with the surface combined with the epoxy group. Silicon oxide of the detection chip is activated, so that silicon hydroxyl is generated on the surface of the detection chip; then, the silicon hydroxyl reacts with 3-glycerol propyl trimethoxy silane, and high-density epoxy distribution is formed on the surface of the detection chip. That is to say, the surface of the detection chip generates the epoxy group connected by the chemical bond through the series of surface chemical reactions, so that the problem that a compound film containing the epoxy group on the detection chip is easy to fall off in the related technology is solved, and the subsequent protein coupling efficiency is improved.
Drawings
FIG. 1 is a flowchart of a modification method for a detection chip according to an embodiment of the present disclosure;
fig. 2 is a schematic structural diagram of a detection chip provided in an embodiment of the present application;
FIG. 3 is a fluorescent image of antibody labeling using the detection chip provided in the embodiments of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings of the embodiments of the present application. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the application without any inventive step, are within the scope of protection of the application.
It is to be noted that materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The dimensions and shapes of the various figures in the drawings are not to scale and are merely intended to illustrate the context of the application. And the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout.
In the related art, a thin film of a compound having an epoxy group or other groups capable of coupling with proteins is deposited or spin-coated on a glass substrate, so as to realize the fabrication of a detection chip for subsequent protein coupling. However, the glass substrate prepared by the fusion process has many defects, so that the compound film has poor adhesion on the glass substrate and is easy to fall off, thereby affecting the protein coupling efficiency.
In view of the above problems in the related art, the embodiments of the present disclosure provide a detection chip, a modification method thereof, and a reaction system.
Specifically, the modification method for detecting a chip provided in the embodiment of the present application, as shown in fig. 1, includes the following steps:
s101, preparing a silicon oxide SiOxThe detection chip of (1) is activated to convert the silicon oxide into silicon hydroxyl, as shown in reaction formula I;
reaction formula I
S102, placing the detection chip with the silicon hydroxyl in an organic solution of 3-glyceryl propyl trimethoxy silane to obtain the detection chip with the surface combined with the epoxy group, wherein the reaction formula II is shown.
Reaction formula II
In the modification method provided by the embodiment of the application, a series of surface chemical reaction operations are performed on the surface of the detection chip, so that the surface of the detection chip generates the epoxy groups connected through chemical bonds, the problem that a compound film containing the epoxy groups on the detection chip is easy to fall off in the related technology is solved, and the subsequent protein coupling efficiency is improved. The microfluidic system is suitable for in vitro diagnosis, drug property screening, cell culture, immunofluorescence detection and the like.
Optionally, in the modification method provided in this embodiment of the present application, in step S101, the detection chip with a silicon hydroxyl group is placed in an organic solution of 3-glyceropropyltrimethoxysilane, so as to obtain a detection chip with an epoxy group bound to the surface, which may be specifically implemented in the following manner:
vertically placing the detection chip with the silicon hydroxyl into an epoxidation fixture according to the fixture structure, injecting 0.5-5% (v/v) of 3-glyceropropyl trimethoxy silane (GPTMS) in toluene solution into the epoxidation fixture, and hermetically soaking for 24-72 h at the temperature of room temperature to 70 ℃.
Specifically, the volume ratio (v/v) of 3-Glyceropropyltrimethoxysilane (GPTMS) in toluene may be 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.; the toluene can be selected from ultra-dry toluene; the soaking temperature is room temperature (generally at about 25 deg.C, such as 16-18 deg.C in winter and 24-26 deg.C in summer), 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C; the soaking time can be 24h, 30h, 36h, 40h, 48h, 60h, 72h and the like.
Preferably, in order to achieve a better epoxidation effect, the detection chip with the silicon hydroxyl groups can be vertically placed into an epoxidation fixture according to a fixture structure, and 3% (v/v) 3-Glyceropropyltrimethoxysilane (GPTMS) ultra-dry toluene solution is injected into the epoxidation fixture, and the epoxy fixture is hermetically soaked for 24 hours at the temperature of 70 ℃.
Optionally, after the step S102 is executed, the reaction solution is poured off, the detection chip is washed twice with deionized water, and then the detection chip is ultrasonically cleaned with deionized water for 5min, so as to remove impurities on the detection chip during the epoxidation process. And then, drying the ultrasonically cleaned detection chip by using nitrogen, and storing the detection chip in the nitrogen atmosphere to finish modification for subsequent use in protein coupling.
Optionally, in the modification method provided in the embodiment of the present application, the step S101 is to activate the detection chip with silicon oxide to convert the silicon oxide into a silicon hydroxyl group, and specifically may be implemented by:
vertically placing a detection chip with silicon oxide into an activation fixture according to the fixture structure, preparing a piranha solution (concentrated sulfuric acid: 30% hydrogen peroxide: 1:3) on site, slowly pouring the solution into the activation fixture without cooling, and stirring in a water bath at 70-90 ℃ for 12-24 h.
Specifically, the water bath stirring temperature is 70 ℃, 72 ℃, 75 ℃, 80 ℃, 83 ℃, 85 ℃, 88 ℃, 90 ℃ and the like, and the water bath stirring time is 12h, 15h, 18h, 20h, 21h, 24h and the like.
Optionally, after the step S101 is performed, the piranha solution is poured out and properly processed, the detection chip is washed twice with deionized water, then the detection chip is ultrasonically cleaned with deionized water for 10min, impurities stained on the surface of the detection chip in the activation process are removed, and finally the detection chip is dried by nitrogen for standby.
Optionally, in the modification method provided in the embodiment of the present application, before performing the step S101 of activating the detection chip having silicon oxide, the following steps may be further performed:
and sequentially adopting acetone, ethanol and deionized water to ultrasonically clean the detection chip with the silicon oxide, and adopting nitrogen to blow the cleaned detection chip for later use.
Specifically, after cutting Corning Eagle glass with a thickness of 0.5mm into a standard glass slide size of 1in × 3in, the glass slide is loaded into a cleaning fixture for pre-cleaning, and the cleaning process flow sequentially comprises the following steps: ultrasonic cleaning with acetone for 10min, ultrasonic cleaning with ethanol for 10min, ultrasonic cleaning with deionized water for 10min, and ultrasonic cleaning with deionized water for 10 min. Thus, other impurities such as grease on the surface of the glass can be cleaned. And after cleaning, drying the detection chip by nitrogen for later use.
Optionally, in the modification method provided in this embodiment of the present application, before performing ultrasonic cleaning on the detection chip with silicon oxide using acetone, the following steps may also be performed:
and pretreating the glass substrate to form the detection chip with silicon oxide.
In the related art, the glass substrate of the detection chip has a certain degree of hydrophobicity, and the solvent contained in the solution to be detected in the biological field is generally water, so that the solution to be detected is not in good contact with the glass substrate, and the marker in the solution to be detected is not favorably combined with the detection chip. The silicon oxide has hydrophilicity for the detection chip that this application provided can realize better and wait to detect the in close contact with of solution, promote detection effect.
Optionally, in the modification method provided in this embodiment of the present application, the step of pretreating the glass substrate to form the detection chip having silicon oxide may be specifically implemented in the following manner:
forming a plurality of spotting platforms on a glass substrate;
and depositing a hydrophilic layer which is 300nm thick and is made of silicon oxide on the layer where each sampling platform is located by adopting a Plasma Enhanced Chemical Vapor Deposition (PECVD) method at the temperature of 390 ℃, etching the hydrophilic layer, and reserving the silicon oxide in the area where each sampling platform is located to obtain the detection chip with the silicon oxide.
The hydrophilic layer made of the silicon oxide material formed by the method has the advantages of good film thickness uniformity, few film layer pinholes, difficult cracking and the like, so that the contact effect of the solution to be detected and the detection chip is better.
It should be noted that the parameters such as time and temperature appearing in the modification process are only illustrative and not limiting.
Based on the same inventive concept, the embodiment of the present application further provides a detection chip, including: the device comprises a glass substrate, a plurality of sample application platforms positioned on the glass substrate and a hydrophilic layer covering each sample application platform, wherein the hydrophilic layer comprises silicon oxide with a chemical modification group; wherein the content of the first and second substances,
the chemical modifying group is obtained by the above modification method provided in the examples of the present application.
Specifically, as shown in fig. 2, a schematic structural diagram of the detection chip provided in the embodiment of the present application is shown. Wherein 201 denotes a lower glass substrate, 202 denotes a spotting platform, 203 denotes silicon oxide, 203' denotes a chemical modification group, 204 denotes a flow guide dam, 205 denotes a hydrophobic layer, 206 denotes an upper glass substrate, 207 denotes a sample inlet, 208 denotes a support member, and 209 denotes a positioning member.
The chemical modification group 203 'is obtained by the above modification method provided in the embodiments of the present application, and thus the chemical modification group 203' has an epoxy group, which can bind to the target antigen or antibody. Specifically, in fig. 3, the lighter colored region (i.e., the approximately circular portion in fig. 3) shows the result of the test of the efficiency of the attachment between the fluorescence-labeled antibody and the epoxy group in the chemical modification group 203'. The results prove that: the detection chip provided by the application has high epoxy group grafting density on the surface, so that when the detection chip is combined with proteins such as antibodies, the detection chip has ultrahigh protein coupling efficiency and ultralow non-specific adsorption.
In the actual immunoassay process, the solution to be detected is injected into the solution flowing space to be detected, which is defined by the lower glass substrate 201, the upper glass substrate 206 and the supporting component 208 together, through the injection port 170. Under the dredging action of the diversion dam 204 and the hydrophobic action of the hydrophobic layer 205, the solution to be detected uniformly and stably flows through the area where each sample application platform 202 is located. The chemical modification group 203 'obtained by the modification method based on the silicon oxide 203 is combined with the target antigen or antibody, so that the marker in the solution to be detected can be combined with the target antigen or antibody on the chemical modification group 203' in the process that the solution to be detected flows through the spotting platform 202, and the marker is fixed on the detection chip. Then, the positioning component 209 cooperates with a separately provided optical detection device to realize the positioning of the detection chip, so as to perform optical detection (for example, fluorescence detection) on the detection chip, thereby obtaining an immunoassay result.
Based on the same inventive concept, the embodiment of the present application further provides a reaction system, including: the above-mentioned detection chip that this application embodiment provided. Because the principle of the reaction system for solving the problems is similar to that of the detection chip, the implementation of the reaction system provided by the embodiment of the present invention can refer to the implementation of the detection chip provided by the embodiment of the present invention, and repeated details are not repeated.
As can be seen from the above description, in the detection chip, the modification method thereof, and the reaction system provided in the embodiment of the present application, silicon hydroxide groups are generated on the surface of the detection chip by activating silicon oxide of the detection chip; then, the silicon hydroxyl reacts with 3-glycerol propyl trimethoxy silane, and high-density epoxy distribution is formed on the surface of the detection chip. That is to say, the surface of the detection chip generates the epoxy group connected by the chemical bond through the series of surface chemical reactions, so that the problem that a compound film containing the epoxy group on the detection chip is easy to fall off in the related technology is solved, and the subsequent protein coupling efficiency is improved. The microfluidic system is suitable for in vitro diagnosis, drug property screening, cell culture, immunofluorescence detection and the like. In addition, the modification method provided by the application is carried out based on a glass substrate, so that the mass production is facilitated, and the cost is effectively reduced. In addition, as can be seen from the above description, the modification method provided by the present application is relatively simple in operation process, and facilitates to improve efficiency.
It should be noted that, the present application describes the process method of the present application through the above examples, but the present application is not limited to the above process steps, i.e., the present application does not mean that the present application has to rely on the above process steps to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitution of selected materials for the present application, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present application.
Claims (10)
1. A modification method for a detection chip is characterized by comprising the following steps:
activating the detection chip with the silicon oxide to convert the silicon oxide into a silicon hydroxyl group;
and (3) placing the detection chip with the silicon hydroxyl in an organic solution of 3-glyceryl propyl trimethoxy silane to obtain the detection chip with the surface combined with an epoxy group.
2. The modification method according to claim 1, wherein the detection chip having the silicon hydroxyl group is placed in an organic solution of 3-glyceropropyltrimethoxysilane to obtain the detection chip having an epoxy group bonded to the surface thereof, and specifically comprises:
and (3) putting the detection chip with the silicon hydroxyl into a toluene solution of 0.5-5% (v/v) 3-glyceropropyl trimethoxy silane, and hermetically soaking for 24-72 h at the temperature of between room temperature and 70 ℃.
3. The modification method according to claim 2, wherein the detection chip having the silicon hydroxyl group is placed in a toluene solution of 0.5% -5% (v/v) 3-glyceropropyltrimethoxysilane, and hermetically soaked for 24-72 h at a temperature of between room temperature and 70 ℃, and specifically comprises:
the detection chip with the silicon hydroxyl group is put into a toluene solution of 3% (v/v) 3-glyceropropyl trimethoxy silane and is hermetically soaked for 24 hours at 70 ℃.
4. The modification method according to claim 1, wherein the activation treatment of the detection chip having silicon oxide to convert the silicon oxide into a silicon hydroxyl group specifically comprises:
putting the detection chip with silicon oxide into the piranha solution, and soaking for 12-24 h at the temperature of 70-90 ℃; the piranha solution is composed of concentrated sulfuric acid and 30% hydrogen peroxide, wherein the volume ratio of the concentrated sulfuric acid to the 30% hydrogen peroxide is 1: 3.
5. A modification method as claimed in claim 1, characterized in that, after each step has been carried out, the detection chip is subjected to the following treatment:
washing the detection chip twice by using deionized water;
and ultrasonically cleaning the washed detection chip in deionized water, and drying the detection chip by using nitrogen for later use.
6. The modification method according to any one of claims 1 to 5, further comprising, before the activation treatment of the detection chip having silicon oxide:
and sequentially adopting acetone, ethanol and deionized water to ultrasonically clean the detection chip with the silicon oxide, and adopting nitrogen to blow the cleaned detection chip for later use.
7. The modification method according to claim 6, further comprising, before said ultrasonic cleaning of said detection chip having silicon oxide with acetone:
and pretreating the glass substrate to form the detection chip with silicon oxide.
8. The modifying method according to claim 7, wherein the pretreating the glass substrate to form the detection chip having silicon oxide specifically comprises:
forming a plurality of spotting platforms on the glass substrate;
and depositing a hydrophilic layer which is 300nm thick and is made of silicon oxide on the layer where the sample application platforms are located by adopting a plasma enhanced chemical vapor deposition method at the temperature of 390 ℃, etching the hydrophilic layer, and reserving the silicon oxide in the area where the sample application platforms are located to obtain the detection chip with the silicon oxide.
9. A detection chip, comprising: the printing device comprises a glass substrate, a plurality of printing platforms positioned on the glass substrate, and a hydrophilic layer covering each printing platform, wherein the hydrophilic layer comprises silicon oxide with a chemical modification group; wherein the content of the first and second substances,
the chemical modification group is obtained by the modification method according to any one of claims 1 to 8.
10. A reaction system comprising the detection chip according to claim 9.
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| CN202010197439.3A CN111266142A (en) | 2020-03-19 | 2020-03-19 | Detection chip, modification method thereof and reaction system |
| US17/631,461 US20220274106A1 (en) | 2020-03-19 | 2021-03-03 | Detection chip and modification method therefor |
| PCT/CN2021/078950 WO2021185087A1 (en) | 2020-03-19 | 2021-03-03 | Detection chip and modification method therefor |
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| US20220274106A1 (en) | 2022-09-01 |
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